Human primary verruca verrucosa endothelial cell model construction method, medicine and application

Abstract

The invention discloses a human primary verrucoid hemangioma endothelial cell model construction method, a medicine and application, human primary verrucoid hemangioma endothelial cells can be successfully obtained, and a solid foundation is laid for screening the medicine for treating human verrucoid hemangioma. The method comprises the following steps: (1) constructing HUVEC cells transfected with MAP3K3 lentivirus; (2) detecting the expression level of the MEKK3 protein through a Western Blot experiment; and (3) separating the human primary verrucoid hemangioma endothelial cells.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a method for constructing an endothelial cell model of human primary verrucous hemangioma, a drug and an application thereof in verrucous vascular malformations. Background Art

[0002] Verrucous venous malformation (VVM) is a rare congenital vascular lesion, which is a superficial vascular keratoma-like vascular malformation composed of capillaries and veins in the dermis and subcutaneous tissue. VVM is often present at birth or in childhood, and is commonly found on one side of the lower limbs, upper limbs, feet and buttocks. VVM lesions appear as irregular dark red plaques with clear borders. With age, the epidermal keratinization gradually worsens, and may be accompanied by bleeding, ulceration and secondary infection. The pathogenesis of VVM is still unclear, and it may be related to somatic missense mutations in MAP3K3. Many studies have detected somatic mutations in the MAP3K3 gene (c.1323C>G, p.Iso441Met) in VVM lesion tissue. Skin hemangioma seriously affects the appearance and image of children, causing them to have a series of psychological disorders such as introversion, autism, inferiority, and extremism. Surgical excision is the first choice for the treatment of VVM, but the recurrence rate after surgery is high, and patients with large-area skin lesions require multiple surgeries. To further reduce the recurrence rate, many treatment methods have been used in combination with surgical resection, such as cryotherapy, electrocautery, laser therapy, etc., but the effects are limited.

[0003] Clinical studies have shown that oral rapamycin (0.8 mg / m 2 , twice a day) can effectively reduce the area of ​​skin lesions. Rapamycin is a low-toxic macrolide antibiotic and an inhibitor of the target of rapamycin (mTOR). Rapamycin acts on the mTOR pathway and forms a complex that inhibits the activation of the mTOR complex, thereby reducing protein synthesis, inducing cell apoptosis, inhibiting cell migration, and reducing the expression of vascular endothelial growth factor. It has a therapeutic effect of anti-angiogenesis and anti-lymphogenesis.

[0004] However, some patients do not respond to rapamycin. Summary of the invention

[0005] In order to overcome the defects of the prior art, the technical problem to be solved by the present invention is to provide a method for constructing an endothelial cell model of primary human verrucous hemangioma, which can successfully obtain primary human verrucous hemangioma endothelial cells and lay a solid foundation for screening drugs for treating human verrucous hemangioma.

[0006] The technical solution of the present invention is: a method for constructing a human primary verrucous hemangioma endothelial cell model, which comprises the following steps:

[0007] (1) Construction of HUVEC cells transfected with MAP3K3 lentivirus: Overexpression control CON lentivirus and overexpression MAP3K3 lentivirus were added to a six-well plate at a multiplicity of infection index of 10. WT , point mutation lentiviral MAP3K3 I441M MAP3K3 gene knockdown control Sh0 lentivirus, MAP3K3 gene knockdown Sh1 lentivirus, MAP3K3 gene knockdown Sh2 lentivirus, MAP3K3 gene knockdown Sh3 lentivirus; remove the virus 24 hours after transfection, add new complete medium and culture for 72 hours, see green fluorescent protein expression, add puromycin at a concentration of 800ng / ml to the culture dish for 48 hours, remove the non-transfected cells, and obtain transfected cells;

[0008] (2) Detect the expression level of MEKK3 protein by Western Blot experiment;

[0009] (3) Isolation of primary human verrucous hemangioma endothelial cells.

[0010] The beneficial effects of the present invention are: human primary verrucous hemangioma endothelial cells can be successfully obtained, laying a solid foundation for screening drugs for treating human verrucous hemangioma.

[0011] Also provided is a drug for preparing a drug for treating human primary verrucous hemangioma, wherein the active ingredients of the drug include rapamycin and ponatinib.

[0012] Also provided is the use of the drug in verrucous vascular malformations, which downregulates the expression level of MAP3K3 protein in human primary verrucous hemangioma endothelial cells and inhibits cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Western Blot results showed that MAP3K3 was overexpressed in WT Lentiviral expression of MAP3K3 with point mutations I441M The expression level of MEKK3 protein in the cells was significantly higher than that in the CON lentivirus transfection group.

[0014] Figure 2 Western Blot results showed that the expression level of MEKK3 protein in cells transfected with MAP3K3 gene knockdown Sh3 lentivirus was significantly lower than that in cells transfected with MAP3K3 gene knockdown control Sh0 lentivirus.

[0015] Figure 3 The results for MTS showed that transfection overexpressed MAP3K3 WT Lentiviral expression of MAP3K3 with point mutations I441M The cell proliferation was significantly higher than that in the CON lentivirus transfection group.

[0016] Figure 4 The MTS results showed that the proliferation of cells transfected with MAP3K3 gene knockdown Sh3 lentivirus was significantly lower than that of cells transfected with MAP3K3 gene knockdown control Sh0 lentivirus.

[0017] Figure 5 Immunofluorescence confirmed that CD31 was expressed on the membrane of isolated primary human verrucous hemangioma endothelial cells.

[0018] Figure 6 The results of the EdU cell proliferation experiment showed that compared with the use of either drug alone, the combined use of rapamycin and ponatinib can significantly inhibit the proliferation of human primary verrucous hemangioma endothelial cells.

[0019] Figure 7 The figure is a flow chart of the method for constructing the human primary verrucous hemangioma endothelial cell model according to the present invention. DETAILED DESCRIPTION

[0020] In order to further optimize the treatment of VVM and improve the treatment efficiency, the inventors searched for many drugs and found that Ponatinib (AP24534) can inhibit the progression of cerebral cavernous malformations in mice by inhibiting the MAP3K3-LKF signaling pathway. Ponatinib can exert anti-angiogenic effects in zebrafish and human umbilical vein endothelial cells by blocking the VEGFR signaling pathway. Ponatinib is currently used to treat adult chronic myeloid leukemia (CML) and "Philadelphia chromosome-positive" (Ph+) acute lymphoblastic leukemia (ALL) that are resistant to the tyrosine kinase inhibitors imatinib, dasatinib or nilotinib due to T315I mutations. There have been no reports on the treatment of VVM with rapamycin combined with ponatinib, so the inventors speculate that the use of rapamycin combined with ponatinib may be a new method for treating VVM.

[0021] The present invention constructs a lentivirus (MAP3K3) that overexpresses MAP3K3 WT ), MAP3K3p.Iso441Met point mutation lentivirus (MAP3K3 I441M ), MAP3K3 gene knockdown virus (Sh1, Sh2, Sh3), Western Blot results showed MAP3K3 I441MThe mutation increased the expression of MEKK3 protein, which was consistent with the trend of overexpression lentivirus. The MAP3K3 gene knockdown lentivirus Sh3 reduced the expression of MEKK3 protein. The MTS results showed that both overexpression and mutation of the MAP3K3 gene promoted cell proliferation, and knockdown of the MAP3K3 gene (Sh3) inhibited cell proliferation. The present invention is the first to use rapamycin combined with ponatinib to treat the human primary verrucous hemangioma endothelial cell model. It was found that rapamycin combined with ponatinib can downregulate the expression level of MAP3K3 protein in human primary verrucous hemangioma endothelial cells and inhibit cell proliferation. This effect is better than the use of rapamycin or ponatinib alone.

[0022] like Figure 7 As shown, the method for constructing the endothelial cell model of primary human verrucous hemangioma comprises the following steps:

[0023] (1) Construction of HUVEC cells transfected with MAP3K3 lentivirus: Overexpression control CON lentivirus, overexpression MAP3K3 lentivirus and 10 lentiviruses were added to a six-well plate at a multiplicity of infection (MOI) of 10. WT , point mutation lentiviral MAP3K3 I441M , MAP3K3 gene knockdown control Sh0 lentivirus, MAP3K3 gene knockdown Sh1 lentivirus, MAP3K3 gene knockdown Sh2 lentivirus, MAP3K3 gene knockdown Sh3 lentivirus; 24 hours after transfection, the virus was removed, and new complete medium was added for 72 hours. When green fluorescent protein (GFP) expression was observed, puromycin was added to the culture dish at a concentration of 800 ng / ml for 48 hours, and the non-transfected cells were removed to obtain transfected cells;

[0024] (2) Detect the expression level of MEKK3 protein by Western Blot experiment;

[0025] (3) Isolation of primary human verrucous hemangioma endothelial cells.

[0026] The beneficial effects of the present invention are: human primary verrucous hemangioma endothelial cells can be successfully obtained, laying a solid foundation for screening drugs for treating human verrucous hemangioma.

[0027] Preferably, the step (2) comprises the following sub-steps:

[0028] (2.1) HUVEC cells transfected with lentivirus were seeded in 96-well plates at a seeding density of 4×10 3 Each well was filled with 100ul complete culture medium, and each group had 5 parallel wells. The cells were cultured in a 37°C, 5% CO2 incubator.

[0029] (2.2) On the 1st, 2nd and 3rd day after cell inoculation, take out the MTS reagent and dissolve it at room temperature, add 10 μl of MTS detection solution to each well, and incubate at 37°C, 5% CO2 in the dark for 2 hours;

[0030] (2.3) Use an ELISA reader to measure the optical density (OD) at 490 nm;

[0031] (2.4) Plot the cell growth curve with time as the horizontal axis and absorbance as the vertical axis, and statistically analyze the differences in absorbance between groups at each time point.

[0032] Preferably, the step (3) comprises the following sub-steps:

[0033] (3.1) Trim the hemangioma tissue into small pieces, rinse, and place in a centrifuge tube containing a neutral proteinase II solution with a final concentration of 2.4 U / ml, and shake and digest at 4°C overnight;

[0034] (3.2) The next day, a single cell suspension was obtained, and the cell pellet was resuspended in ECM complete medium. The cells were seeded in a culture dish containing ECM complete medium, and the cell adhesion was observed under a microscope the next day;

[0035] (3.3) When the cell confluence is 60%, add trypsin to the culture dish for digestion. After 90% of the cells shrink and become round, add an equal amount of complete culture medium to terminate the digestion. Centrifuge and add PBS to the centrifuge tube to resuspend the cell pellet. Centrifuge and discard the supernatant.

[0036] (3.4) Incubate CD31 magnetic beads at 4°C for 15 minutes, resuspend the cell pellet with ECM complete medium, drip the cell suspension into the sorting column, and elute the CD31-negative cells with ECM complete medium. Immediately drip ECM complete medium after the cells are all washed off. Repeat the elution three times, remove the sorting column from the sorter, drip ECM complete medium into the sorting column, insert the plunger to push out the CD31-positive cells; after removing the plunger, drip ECM complete medium again to push out the CD31-positive cells. The obtained CD31-positive cells are human primary verrucous hemangioma endothelial cells.

[0037] Preferably, the step (3) further comprises a sub-step (3.5): culturing the CD31 positive cells, observing under a microscope that the magnetic beads on the cell surface have been washed away, continuing to culture until the cells are fully grown, then digesting and collecting the cells to prepare a cell suspension, and culturing the cells at a concentration of about 1×10 5 / ml were inoculated in confocal culture dishes and cultured for 24 hours; the culture medium was discarded, the cells were rinsed twice with PBS, the cells were fixed with -20℃ precooled methanol at 4℃ for 15 minutes, and the cells were washed three times with PBS, each time for 5 minutes; 5% BSA was blocked at room temperature for 1 hour, CD31 primary antibody was incubated at 4℃ overnight; the cells were washed three times with PBS for 5 minutes each; the fluorescent secondary antibody was incubated at room temperature in the dark for 1 hour, and the cells were washed three times with PBS for 5 minutes each; 1mg / ml ready-to-use DAPI was incubated at room temperature in the dark for 3 minutes, and the cells were washed three times with PBS for 5 minutes each; the percentage of CD31-positive cells was observed and analyzed using a confocal microscope.

[0038] Preferably, in the step (3.1), in the clean bench of the laboratory, the hemangioma specimen is taken out and placed in a 10 cm culture dish, washed three times with sterile PBS to wash away residual blood and culture medium, transferred to a new culture dish, and the excess skin tissue and fat tissue are carefully trimmed with sterile tissue scissors to completely separate the hemangioma tissue, and the hemangioma tissue is placed in a new culture dish, and then rinsed twice with PBS containing 1% double antibody, and the hemangioma tissue is trimmed into small pieces in the culture dish with tissue scissors, and then rinsed twice with PBS containing 1% double antibody, and the tissue blocks are placed in a centrifuge tube containing a neutral protease II solution with a final concentration of 2.4 U / ml, and shaken and digested overnight at 4°C.

[0039] Preferably, in step (3.2), the next day, after centrifugation at 1000 rpm for 5 min, the supernatant was discarded, the tissue block was washed twice with PBS, the tissue block was placed in a culture dish, DMEM culture medium was added to keep it moist, and the tissue block was cut into 1 mm pieces with ophthalmic scissors. 3 The cells were transferred to a centrifuge tube containing a final concentration of 2mg / ml collagenase type I and 50ug / ml DNase solution, and digested in a 37℃ water bath for 2 hours; the mixed digestion solution was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended with 2ml DMEM complete medium. The cell suspension was filtered through a 150-mesh sterile filter and then filtered through a 30um pore size sterile filter membrane to obtain a single-cell suspension; the filtrate was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended with 1ml ECM complete medium. The cells were inoculated in a culture dish containing ECM complete medium, and the cell adhesion was observed under a microscope the next day.

[0040] Preferably, in the step (3.3), when the cell confluence is 60%, the culture medium is aspirated and the cells are rinsed twice with PBS, trypsin is added to the culture dish for digestion, and after observing that 90% of the cells shrink and become round, an equal amount of complete culture medium is added to terminate the digestion; the cell suspension is collected into a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant is aspirated and the cell pellet is washed with PBS, the supernatant is aspirated and the cell pellet is washed with PBS, the supernatant is aspirated and the cell pellet is resuspended by adding 1 ml of PBS to the centrifuge tube, and the cell pellet is transferred to a new 15 ml centrifuge tube, and the supernatant is aspirated and the cell pellet is centrifuged at 1000 rpm for 5 minutes.

[0041] Preferably, in the step (3.4), 60ul ECM complete medium, 20ul FcR blocking solution, and 20ul CD31 immunomagnetic beads are added to the centrifuge tube, and gently shaken and incubated at 4°C for 15min; 1ml ECM complete medium is added to the centrifuge tube and mixed, centrifuged at 1000rpm for 3 minutes, the supernatant is discarded, and the cell pellet is resuspended in 1ml ECM complete medium; the magnetic stand is disinfected and placed on a clean bench, the MACS sorting column is placed in the MACS sorter, and the sorting column is rinsed with 3ml ECM complete medium; the cell suspension is dripped into the sorting column, and the CD31-negative cells are eluted with 3ml ECM complete medium, and after dripping, 3ml ECM complete medium is dripped, and the elution is repeated 3 times; the sorting column is removed from the sorter, 5ml ECM complete medium is dripped into the sorting column, and the plunger is inserted to push out the CD31-positive cells; after the plug is pulled out, 1ml ECM complete culture medium was used to push out CD31-positive cells, and the process was repeated 3 times. The obtained CD31-positive cells were human primary verrucous hemangioma endothelial cells.

[0042] Also provided is a drug for preparing a drug for treating human primary verrucous hemangioma, wherein the active ingredients of the drug include rapamycin and ponatinib.

[0043] Also provided is the use of the drug in verrucous vascular malformations, which downregulates the expression level of MAP3K3 protein in human primary verrucous hemangioma endothelial cells and inhibits cell proliferation.

[0044] The embodiments of the present invention are described in detail below.

[0045] The following verification shows that rapamycin combined with ponatinib can inhibit the proliferation of human primary verrucous hemangioma endothelial cells.

[0046] 1) Digest the cells in the logarithmic growth phase and resuspend them in complete medium to a density of 2×10 5 / ml, inoculated in a confocal culture dish, and cultured overnight in a 37°C constant temperature cell culture incubator. The next day, the serum-free medium was replaced with a 37°C constant temperature cell culture incubator for 24 hours. The next day, a culture medium containing 1% fetal bovine serum was used to prepare a final concentration of 20 μM rapamycin, 1 μM ponatinib, and a combination of experimental culture medium, and human primary verrucous hemangioma endothelial cells were treated for 24 hours.

[0047] 2) Dilute EdU to 20 μM with medium containing 1% fetal bovine serum, add 1 ml of the diluted EdU working solution to each dish to make the final concentration 10 μM, continue incubation at 37°C for 3 hours, remove the cell culture medium, wash twice with 3% BSA, 3 minutes each time. Remove the washing solution, add 500 μl of 4% paraformaldehyde to each well to fix the cells at room temperature for 15 minutes. Remove the fixative, wash three times with 3% BSA, 3 minutes each time. Remove the washing solution, add 500 μl of permeabilization solution (0.3% TritonX-100 in PBS) to each well and incubate at room temperature for 15 minutes. Remove the permeabilization solution, wash twice with 3% BSA, 3 minutes each time. Prepare the reaction solution according to the instructions of the kit, add 200 μl of the reaction solution to each dish, and incubate at room temperature for 30 minutes in the dark. Remove the reaction solution, wash three times with 3% BSA, 3 minutes each time. Remove the washing solution, add 100 μl 1× Hoechst33342 to each dish, and stain for 10 minutes at room temperature in the dark. Remove Hoechst and wash 3 times with 3% BSA for 3 minutes each time. Observe EdU-labeled and unlabeled cells using a confocal microscope, take pictures and count. EdU labeling indicates that the cells are in a proliferative state. When rapamycin and ponatinib are used in combination, the red fluorescence of EdU labeling is significantly weakened, indicating that DNA synthesis is significantly inhibited.

[0048] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a human primary verrucous hemangioma endothelial cell model, characterized in that: It includes the following steps: (1) Construction of HUVEC cells transfected with MAP3K3 lentivirus: Overexpression control CON lentivirus and overexpression MAP3K3 lentivirus were added to a six-well plate at a multiplicity of infection index of 10. WT , point mutation lentiviral MAP3K3 I441M MAP3K3 gene knockdown control Sh0 lentivirus, MAP3K3 gene knockdown Sh1 lentivirus, MAP3K3 gene knockdown Sh2 lentivirus, MAP3K3 gene knockdown Sh3 lentivirus; remove the virus 24 hours after transfection, add new complete medium and culture for 72 hours, see green fluorescent protein expression, add puromycin at a concentration of 800ng / ml to the culture dish for 48 hours, remove the non-transfected cells, and obtain transfected cells; (2) Detect the expression level of MEKK3 protein by Western Blot experiment; (3) Isolation of primary human verrucous hemangioma endothelial cells.

2. The method for constructing a human primary verrucous hemangioma endothelial cell model according to claim 1, characterized in that: The step (2) comprises the following sub-steps: (2.1) HUVEC cells transfected with lentivirus were seeded in 96-well plates at a seeding density of 4×10 3 Each well was filled with 100ul complete culture medium, and each group had 5 parallel wells. The cells were cultured in a 37°C, 5% CO2 incubator. (2.2) On the 1st, 2nd and 3rd day after cell inoculation, take out the MTS reagent and dissolve it at room temperature, add 10 μl of MTS detection solution to each well, and incubate at 37°C, 5% CO2 in the dark for 2 hours; (2.3) Use an ELISA reader to measure the optical density at 490 nm; (2.4) Plot the cell growth curve with time as the horizontal axis and absorbance as the vertical axis, and statistically analyze the differences in absorbance between groups at each time point.

3. The method for constructing a primary human verrucous hemangioma endothelial cell model according to claim 2, characterized in that: The step (3) comprises the following sub-steps: (3.1) Trim the hemangioma tissue into small pieces, rinse, and place in a centrifuge tube containing a neutral proteinase II solution with a final concentration of 2.4 U / ml, and shake and digest at 4°C overnight; (3.2) The next day, a single cell suspension was obtained, and the cell pellet was resuspended in ECM complete medium. The cells were seeded in a culture dish containing ECM complete medium, and the cell adhesion was observed under a microscope the next day; (3.3) When the cell confluence is 60%, add trypsin to the culture dish for digestion. After 90% of the cells shrink and become round, add an equal amount of complete culture medium to terminate the digestion. Centrifuge and add PBS to the centrifuge tube to resuspend the cell pellet. Centrifuge and discard the supernatant. (3.4) Incubate the CD31 magnetic beads at 4°C for 15 minutes, resuspend the cell pellet with ECM complete medium, drip the cell suspension into the sorting column, and elute the CD31-negative cells with ECM complete medium. Immediately drip ECM complete medium after the cells are all washed off. Repeat the elution three times, remove the sorting column from the sorter, drip ECM complete medium into the sorting column, insert the plunger to push out the CD31-positive cells; after removing the plunger, drip ECM complete medium again to push out the CD31-positive cells. The obtained CD31-positive cells are primary human verrucous hemangioma endothelial cells.

4. The method for constructing a human primary verrucous hemangioma endothelial cell model according to claim 3, characterized in that: The step (3) further comprises a sub-step (3.5): culturing the CD31 positive cells, observing under a microscope that the magnetic beads on the cell surface have been washed away, continuing to culture until the cells are fully grown, then digesting and collecting the cells to prepare a cell suspension at a concentration of about 1×10 5 / ml were inoculated in confocal culture dishes and cultured for 24 hours; the culture medium was discarded, the cells were rinsed twice with PBS, the cells were fixed with -20℃ precooled methanol at 4℃ for 15 minutes, and the cells were washed three times with PBS, each time for 5 minutes; 5% BSA was blocked at room temperature for 1 hour, CD31 primary antibody was incubated at 4℃ overnight; the cells were washed three times with PBS for 5 minutes each; the fluorescent secondary antibody was incubated at room temperature in the dark for 1 hour, and the cells were washed three times with PBS for 5 minutes each; 1mg / ml ready-to-use DAPI was incubated at room temperature in the dark for 3 minutes, and the cells were washed three times with PBS for 5 minutes each; the percentage of CD31-positive cells was observed and analyzed using a confocal microscope.

5. The method for constructing a human primary verrucous hemangioma endothelial cell model according to claim 3, characterized in that: In the step (3.1), in the clean bench of the laboratory, the hemangioma specimen is taken out and placed in a 10 cm culture dish, washed three times with sterile PBS to wash away residual blood and culture medium, transferred to a new culture dish, and the excess skin tissue and fat tissue are carefully trimmed with sterile tissue scissors to completely separate the hemangioma tissue, and the hemangioma tissue is placed in a new culture dish, and then rinsed twice with PBS containing 1% double antibody, and the hemangioma tissue is trimmed into small pieces in the culture dish with tissue scissors, and then rinsed twice with PBS containing 1% double antibody, and the tissue block is placed in a centrifuge tube containing a neutral protease II solution with a final concentration of 2.4 U / ml, and shaken and digested overnight at 4°C.

6. The method for constructing a primary human verrucous hemangioma endothelial cell model according to claim 5, characterized in that: In the step (3.2), the next day, after centrifugation at 1000 rpm for 5 min, the supernatant was discarded, the tissue blocks were washed twice with PBS, the tissue blocks were placed in a culture dish, DMEM culture medium was added to keep them moist, and the tissue blocks were cut into 1 mm pieces with ophthalmic scissors. 3 The cells were transferred to a centrifuge tube containing a final concentration of 2mg / ml collagenase type I and 50ug / ml DNase solution, and digested in a 37℃ water bath for 2 hours; the mixed digestion solution was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended with 2ml DMEM complete medium. The cell suspension was filtered through a 150-mesh sterile filter and then filtered through a 30um pore size sterile filter membrane to obtain a single-cell suspension; the filtrate was centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended with 1ml ECM complete medium. The cells were inoculated in a culture dish containing ECM complete medium, and the cell adhesion was observed under a microscope the next day.

7. The method for constructing a primary human verrucous hemangioma endothelial cell model according to claim 6, characterized in that: In the step (3.3), when the cell confluence is 60%, the culture medium is aspirated and the cells are rinsed twice with PBS, trypsin is added to the culture dish for digestion, and after observing that 90% of the cells shrink and become round, an equal amount of complete culture medium is added to terminate the digestion; the cell suspension is collected into a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the supernatant is aspirated and the cell pellet is washed with PBS, the supernatant is aspirated and the cell pellet is washed with PBS, the supernatant is aspirated and the cell pellet is resuspended by adding 1 ml of PBS to the centrifuge tube, and the cell pellet is transferred to a new 15 ml centrifuge tube, and the supernatant is aspirated and the cell pellet is centrifuged at 1000 rpm for 5 minutes.

8. The method for constructing a primary human verrucous hemangioma endothelial cell model according to claim 7, characterized in that: In the step (3.4), 60ul ECM complete medium, 20ul FcR blocking solution, and 20ul CD31 immunomagnetic beads were added to the centrifuge tube, and the mixture was gently shaken and incubated at 4°C for 15min; 1ml ECM complete medium was added to the centrifuge tube and mixed, and the mixture was centrifuged at 1000rpm for 3 minutes, the supernatant was discarded, and the cell pellet was resuspended in 1ml ECM complete medium; the magnetic stand was disinfected and placed on a clean bench, the MACS sorting column was placed in the MACS sorter, and the sorting column was rinsed with 3ml ECM complete medium; the cell suspension was dripped into the sorting column, and the CD31-negative cells were eluted with 3ml ECM complete medium, and after the cells were dripped clean, 3ml ECM complete medium was dripped into the sorting column, and the elution was repeated 3 times; the sorting column was removed from the sorter, 5ml ECM complete medium was dripped into the sorting column, and the plunger was inserted to push out the CD31-positive cells; after the plug was removed, 1ml ECM complete culture medium was used to push out CD31-positive cells, and the process was repeated 3 times. The obtained CD31-positive cells were human primary verrucous hemangioma endothelial cells.

9. A drug for the treatment of primary human verrucous hemangioma, characterized in that: Its active ingredients include rapamycin and ponatinib.

10. Use of the drug according to claim 9 for treating verrucous vascular malformations, characterized in that: It down-regulates the expression level of MAP3K3 protein in human primary verrucous hemangioma endothelial cells and inhibits cell proliferation.

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